The currently used live poliomyelitis vaccine developed by Sabin (Sabin, A. B. Ann N.Y. Acad. Sci. 61 :924-938 (1955); Sabin, A. B., PASB Sc. Pub. 44: 14-33 (1959); Sabin, A. B., J. Am. Med. Assoc. 194:130-134 (1965)) comprises three attenuated viral strains, corresponding to poliovirus type 1, type 2 and type 3. These vaccines, although very safe and effective, are nevertheless genetically unstable and prone to reversion to neurovirulence. Therefore, each new batch of vaccine requires vigorous safety testing. The only test which has been used for neurovirulence testing of vaccine lots is performed by intrathalamic and/or intraspinal inoculation of monkeys with subsequent pathological evaluation of developing lesions. This test is very expensive, slow and its results may vary since individual responses of monkeys to the inoculum may differ significantly. The same problems are shared by animal safety tests used for other live attenuated vaccines.
Type 3 poliovirus vaccines have been most frequently associated with vaccine-associated cases of polyomyelitis and require the most rigorous animal testing. Studies on type 3 viruses excreted by healthy children after ingestion of the trivalent oral polio vaccine (OPV) regularly showed a change in the nucleotide at position 472 from uridine (U), found in the genome of the type 3 vaccine strain, to cytosine (C), found in wild type strains (Stanway et al, Proc. Natl. Acad. Sci. USA, 81:1539-1543 (1984); Evans et al, Nature, 314:548-550 (1985) and Westrop et al, J. Virol., 63:1338-1344 (1989)). The same mutation was shown to be present in isolates from cases of vaccine-associated disease (Almond, Ann. Rev. Microbiol., 41:153-180 (1987)). This mutation was shown to result in a quantitative increase in histologic lesions scores produced in monkeys after intraspinal inoculation of the virus (Evans et al, Nature, 314:548-550 (1985) and Westrop et al, J. Virol., 63:1338-1344 (1989)).
Sequence changes in poliovirus RNA have been previously analyzed by direct sequencing of viral RNA (Evans et al, Nature, 314:548-550 (1985) and Weeks-Levy et al, Vaccines, 88:223-227, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)), a method which is not particularly sensitive for detection of sequence variants present at low abundance. This approach failed to reveal mutants at position 472 in type 3 poliovirus vaccine lots (Weeks-Levy et al, Vaccines, 88:223-227, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)).